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Abstract

Background

It has been speculated that the biostimulatory effect of Low Level Laser Therapy could
cause undesirable enhancement of tumor growth in neoplastic diseases. The aim of the
present study is to analyze the behavior of melanoma cells (B16F10) in vitro and the in vivo development of melanoma in mice after laser irradiation.

Methods

We performed a controlled in vitro study on B16F10 melanoma cells to investigate cell viability and cell cycle changes
by the Tripan Blue, MTT and cell quest histogram tests at 24, 48 and 72 h post irradiation.
The in vivo mouse model (male Balb C, n = 21) of melanoma was used to analyze tumor volume and
histological characteristics. Laser irradiation was performed three times (once a
day for three consecutive days) with a 660 nm 50 mW CW laser, beam spot size 2 mm2, irradiance 2.5 W/cm2 and irradiation times of 60s (dose 150 J/cm2) and 420s (dose 1050 J/cm2) respectively.

Results

There were no statistically significant differences between the in vitro groups, except for an increase in the hypodiploid melanoma cells (8.48 ± 1.40% and
4.26 ± 0.60%) at 72 h post-irradiation. This cancer-protective effect was not reproduced
in the in vivo experiment where outcome measures for the 150 J/cm2 dose group were not significantly different from controls. For the 1050 J/cm2 dose group, there were significant increases in tumor volume, blood vessels and cell
abnormalities compared to the other groups.

Conclusion

LLLT Irradiation should be avoided over melanomas as the combination of high irradiance
(2.5 W/cm2) and high dose (1050 J/cm2) significantly increases melanoma tumor growth in vivo.

Background

Malignant melanoma represents a burden to modern society and requires considerable
efforts in terms of health service utilization. The incidence is increasing worldwide
and in the Netherlands the prevalence is currently 16.1/100,000 with a mortality rate
of 3.0/100,000[1].

Low level laser therapy (LLLT) has gained increasing popularity as a treatment for
soft tissue injuries and joint conditions. It is applied transcutaneously with typical
irradiances being 10 mW/cm2 - 5,000 mW/cm2, treatments times being in the range of 10 seconds - 2 minutes, with total energy
delivered of 1 - 4 Joules(J)/cm2 per point when targeting joints, tendons and muscles. The cellular proliferative potential
of LLLT irradiation has attracted some negative speculation that this could also increase
tumor growth in neoplasic diseases. Previous studies of LLLT irradiation of tumor
cells in vitro have generated conflicting research data across a range of cultivated
tumor cell lines and irradiation parameters [2-11] but there have been relatively few in vivo studies published [12,13]. In vivo studies are essential for the study of disease development and should be the main
tool for studying the behavior of tumor cells. The complexity of the multi-cellular
environment in an ongoing disease makes it hard to predict tumor behavior and cell
culture studies alone are inadequate to for assessment of tumor responses.

Increases in cell proliferation and collagen biosynthesis after LLLT in wound healing
improvement has already been observed in the pioneer work of Mester et al. [14]. The following decades were marked by a large quantity of research articles in LLLT.
A better understanding of laser light modulatory mechanisms was obtained, but this
effort also yielded conflicting results. There is a shortage of evidence about the
effects of LLLT in malignant conditions such as melanoma. The complete biochemical
mechanisms of cell proliferation after LLLT irradiation are still uncertain and we
believe there is a need to study the effects of LLLT on tumor growth in suitable cell
and animal models.

The aim of the present work is to study the effect of LLLT irradiation both in vitro and in vivo. For this purpose we decided to study cell viability and cell cycle changes in melanoma
cells (B16F10) in vitro, and their behavior when injected subcutaneously into Balb C mice in vivo.

Methods

All the experimental procedures were submitted to and approved by the Ethical Committee
at the Cruzeiro do Sul University.

In vitro laser irradiation

B16F10 cells were irradiated a total of three times (once a day for three consecutive
days) in a 96 well culture plate for the MTT method and in a 12 well plate for Trypan
blue and cytometric assays. Irradiation was performed with a 660 nm, 50 mW Continuous
Wave (CW) laser, beam spot size 2 mm2, irradiance 2.5 W/cm2 (Quasar Medical - Dentoflex, São Paulo, Brasil). The seeded wells were spaced 5 cm
apart in all directions and a thin aluminum sheet was placed halfway (2.5 cm) between
them to prevent unintentional light scattering between the wells. The wells were randomly
divided into a control group which received no irradiation, and a treatment group
which received an LLLT dose of 150 J/cm2 with an irradiance of 2.5 W/cm2 for 60 seconds (3J), while a second group received sessions with an LLLT dose of 1050
J/cm2 with an irradiance of 2.5 W/cm2 for 420 seconds (21J). Total energy delivered after all three sessions was 9J and
63J respectively in the irradiated groups. A support device held the LLLT emission
tip perpendicular to and 2 mm distant from the culture media. Irradiation was carefully
timed and carried out in a dark laminar flux hood.

Animals

The animals were isogenic male Balb C mice (n = 21), which were randomized into one
of three groups; a control group (n = 7), a "low" dose group (n = 7) and a "high"
dose group (n = 7). The mice were injected subcutaneously with a suspension of 2 ×
106 B16F10 melanoma cells.

In vivo laser irradiation

After fifteen days of tumor growth the animals were irradiated three times (once a
day for three consecutive days) at the site of the injected melanoma cells with the
same laser and laser parameters as used in the in vitro study. Irradiation was performed with a 660 nm 660 nm, 50 mW Continuous Wave (CW)
laser, beam spot size 2 mm2, irradiance 2.5 W/cm2 (Quasar Medical - Dentoflex, São Paulo, Brasil). Control Group: Received no irradiation
Group 1: Received three LLLT sessions (once a day for three consecutive days) each
of 60 seconds with a dose of 150 J/cm2, (energy delivered per session was 3J, total energy delivered after three sessions
was 9J) Group 2: Received three sessions (once a day for three consecutive days) each
of 420 seconds with a dose of 1050 J/cm2, (energy delivered per session was 21J, and total energy delivered after three sessions
was 63J).

Outcome measures in vitro

Cell viability and cell changes were determined by MTT method and Trypan blue exclusion
tests (B16F10). Cells were seeded at a density of 1 × 106 cells/Cm2 (B16F10). At the end of the experiment, cells were treated with trypsin (0.05% trypsin
in 0.02% EDTA) and washed 3 times with PBS, fixed in 70% ethanol, and stained with
propidium iodide (PI) 50 mg/10 uL final concentration, these can distinguish hypodiploid
(non-viable or dead cells) from diploid (viable) cells, for 30 min in the dark. All
analyses were done using a FACScalibur flow cytometer (Becton Dickinson, San Jose,
CA). The red fluorescence of PI was collected through a 585/42-nm band-pass filter,
and the fluorescence signals were measured in a linear scale of 1024 channels. For
each sample, at least 10,000 events were acquired and the data were analyzed using
appropriate software (CELLQuest, Becton Dickinson, San Jose, CA). Cell viability was
assessed by counting adherent and non-adherent cells and measured by the cellular
permeability to propidium iodide. Cells in S/G2/M (proliferating) and G0/G1 phases, and hypodiploid cells (cells under death process) were analyzed.

Outcome measures in vivo

Tumor cell growth area was estimated measuring length and width with a paquimeter
device and using the formula: volume = length × width2 π div 6. Histological tumor analysis was performed after tumor volume measurements.
Animals were anaesthetized with inhaled halothane and sacrificed by cervical dislocation.
Tumor mass was immediately removed and immersed in a 4% phosphate buffered paraformaldehide
solution for 48 h. Specimens were dehydratated and embedded in paraffin prior to the
5 μm microtome sections. Histological sections were collected on glass slides and
hematoxylin-eosin stained. Analysis and photographs were carried out in a Nikon-YS100
photomicroscope.

Statistical analysis

The obtained data were first plotted for analysis of normal distribution, and statistical
analysis was then performed with parametric tests if the data were normally distributed.
The statistical level of significance was set at P < 0.05, and significance was tested
statistically by an ANOVA-test. The mean values and its standard error (SE) were calculated,
and differences between control group data and the irradiated group data were tested
statistically with Bonferroni's test.

Results

In vitro experiments

The Trypan Blue dye exclusion test showed no statistical differences in proliferation
or cell death numbers among irradiated groups and control group in the different times
analyzed (Figure 1).

There was statistically a significant difference (p < 0.05) in hypodiploid cells (possible
cell death) at 72 h between the irradiated and control groups (8.48 ± 1.40% and 4.26
± 0.60%). The increase in apoptosis was most prominent in the low dose 150 J/cm2 group (Figure 6).

In vivo experiments

15 days after the B16F10 cell injections all the animals presented average tumor mass
volume of 0.12 ± 0.04 cm3. The increase of the tumor mass volume of control and irradiated groups are shown
in Figure 7.

Figure 7.Graphic showing melanoma tumor growth in the three groups until the 10th day after irradiation.

At the 10th day, the tumor mass volume was significantly higher in the 1050 J/cm2 group when compared to the 150 J/cm2 and the control group. No significant difference in tumor volume was observed between
the 150 J/cm2 and the control group (Figure 8).

Figure 8.Graphic showing the percentage of volume growth on the 10th day after irradiation of the three groups. * = p < 0.05 when compared to the control group indicated by One Way ANOVA and Bonferroni's
Multiple Comparison Test (*P < 0,05).

The macroscopic appearance of dissected tumor differed between the 1050 J/cm2 group and the two other groups. In addition to a marked increase of the volume of
this group, the connective tissue of the capsule appeared sticky to the tumor mass
and to the adjacent muscle tissue. A greater number of blood vessels were also observed
(Figure 9)

Histological sections of the control group revealed a dense mass of melanin producing
melanoma cells invaded by lymphocytes, plasma cells and macrophages. A rich vascular
bed filled with leukocytes and red blood cells can be observed. Some restricted areas
of necrotic tissue were also present. In the connective tissue of the capsule, immunological
cells spread through thin collagen fibers and edema areas (Figure 10).

In the histological sections of the 150 J/cm2 group, immune cells were less frequent in the tumor mass, and large blood vessels
were filled with leukocytes and red blood cells. Necrotic areas were slightly larger
compared to the control group. The connective tissue of the capsule had fewer immune
cells in a greater area of thin fibers of collagen (Figure 11).

Histological sections of the 1050 J/cm2 group showed remarkably atypical melanoma cells. Nuclei were of various sizes and
shapes, and apoptotic figures and the frequency of mitotic cells were high. Necrotic
areas were more common and extensive compared to the other groups. Immune cells were
observed in greater numbers in the tumor mass and in the highly vascular capsule (Figure
12).

Discussion

In the present paper we have investigated the effects of LLLT on malignant melanoma,
in vitro and in vivo. The question of a potential unwanted proliferative effect of low-level laser irradiation,
has been raised by some authors [15,16]. We observed that laser irradiation with a low LLLT dose of 150 J/cm2 presented opposite effects when applied to each distinct situation. In the cultured
melanoma cells, we found that the two LLLT doses presented a non-significant effect
on tumor cells or even an inhibitory effect of cancer cell proliferation through increased
apoptosis. In the in vivo experiment the low dose (150 J/cm2) was not inducing any changes in the cancer cell behavior. However, the high dose
(1050 J/cm2) showed a significant increase in tumor mass volume and considerable histological
alterations which indicate a worsening of the cancer. The results have several implications
for research and clinical practice.

Cell culture is an important method for studying basic biological processes and to
understand the possible cell reactions to treatments. Many kinds of tumor cell lines
have been studied, ranging from carcinomas to sarcomas and myelomas [3,4,6,8,10,17]. We chose B16F10 melanoma cell line because it's a pigmented, highly aggressive and
invasive tumor [18]. Our results of non-significant LLLT effects in the in vitro tests of cell viability are in accordance with the largely non-significant findings
of other authors [9].

Our cell cycle analysis with flow cytometry method indicated a significant increase
in cell death in 72 h of the 1050 J/cm2 group. Some authors have previously found increased cell death in vitro after LLLT irradiation. LLLT fluences higher than 6 J/cm2 seemed to increase cell death in melanoma cell lines (G361, LD50 and SKmel-23), and
especially in melanin producing cells [19]. There seems to be an inverse relationship between laser fluence and melanoma cell
growth in culture [11]. Other authors have reported an increase in G0/G1 phase of the cell cycle using HTB66 melanoma cell line [2], but our results did not support this finding. One important aspect of our findings
is the discrepancy between the in vitro and in vivo experiments. It seems necessary to be careful in generalizing in vitro results, as cell-matrix interactions and cell behavior in the complex environment
of tissues may produce unexpected reactions.

Our results demonstrated a significant tumor growth when the animals were irradiated
with the high dose of 1050 J/cm2. This finding is in line with observations of enhanced Ehrlich ascites tumor growth
after laser irradiation which have been reported in an early paper on LLLT [12]. However it seems that typical LLLT doses ranging from 1 - 4 Joules have no influence
on tumor growth, or rather they can inhibit it in implanted glioma in mice [13].

Histological data also revealed that important differences in cell morphology were
induced by high doses of laser irradiation. The immune cells (lymphocytes, plasma
cells and macrophages) increased in the group irradiated with the high dose of 1050
J/cm2. This group also presented significant areas of necrosis, a high number of atypical
cells and an increase in the number of blood vessels.

Many factors may contribute to tumor growth and most of them can be modulated by laser
irradiation, for instance: low-level laser can enhance angiogenesis [20-22], growth factor synthesis [23-25], inflammatory metabolites [26] as well as modulate immunological cells and inflammation [27-29].

Conclusion

LLLT administered by a dose of 150 J/cm2 appears safe with only minor effects on B16F10 melanoma cells proliferation in vitro and no significant effect on tumor growth in vivo. However, a high irradiance (2.5 W/cm2) combined with high dose of 1050 J/cm2, can stimulate melanoma tumor growth with distinct histological features in vivo. Further studies are necessary to elucidate the main factors that are responsible
for the different behaviors on tumor cells in response to laser light, and to determine
laser irradiance and energy thresholds for stimulation of abnormal melanoma cell behavior.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

LF carried out melanoma injections in mice and histological analysis, JSSL and DAM
carried out melanoma cell culture Trypan Blue dye exclusion test and MTT colorimetric
test, GMF carried out of cell cycle analysis by flow cytometry and statistics, SCP
and JMB were involved in drafting the manuscript and analysis, RABLM and RJB were
involved in revising it critically and gave the final approval of the version to be
published.

All authors have read and approved the final manuscript.

Acknowledgements

This study was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
07/59124-0. and Cruzeiro do Sul University.